During radiation therapy of human tumors the majority of DNA lesions originate from the radiolysis of water in the vicinity of the DNA molecule. These lesions can block DNA replication, which ultimately can lead to cell death and therapeutic efficacy. Alternatively, DNA damage can be bypassed resulting in mutations and the possibility of secondary tumors at the site of treatment by ionizing radiation. Thus it is the interaction between a DNA polymerase and a DNA lesion that ultimately determines the fate of irradiated cells. In order to maximize therapeutic gain and minimize the risk of secondary tumors at the treatment site it is imperative to have a solid mechanistic understanding of the interactions between a human DNA polymerase and a radiation-induced DNA lesion. We propose to address this central issue by studying two types of human DNA polymerases, replicative and specialized, in the context of ionization-induced lesions such as thymine glycol and abasic sites. DNA polymerase ? is a high-fidelity enzyme that plays a crucial role in DNA replication and repair. Pol ? is only somewhat proficient at inserting and extending past abasic sites and is unable to bypass Tg. In contrast, two specialized low-fidelity enzymes, DNA polymerases ? and ?, are much more efficient at extending past these DNA lesions generated by ionizing radiation. We will use a combination of biochemical and structural methods to uncover the mechanisms underpinning replication block or lesion bypass in the context of a replicative or bypass polymerase, with the ultimate goal to design compounds that inhibit pol ?, a polymerase that is overexpressed in breast cancer tumors.